Hyperhomocysteinemia is reportedly a prevalent, independent and graded risk factor for all major forms of vascular disease and thrombosis, and more recently it has been associated with neural tube defects and Alzheimer's disease. Betaine-homocysteine methyltransferase (BHMT) is proposed to have a major role in the regulation of blood homocysteine (Hcy) levels. The physical and mechanistic properties of BHMT and the extent to which variation at the BHMT locus contributes to the incidence of hyperhomocysteinemia represent major gaps in our understanding of Hcy metabolism in both normal and pathological states. Our long-term goal is to elucidate the role BHMT has in regulating Hcy levels in humans. The objective of this application is to characterize further the physical and mechanistic properties of human BHMT, and to elucidate how changes in redox status, including changes in intracellular thiols, hydrogen peroxide (H2O2) and nitric oxide NO) have on its activity. Our central hypothesis is that BHMT is required for normal Hcy netabolism and that drugs or pathological states that increase oxidative stress will inhibit or nactivate BHMT and cause hyperhomocysteinemia. The specific aims of this proposal are (1) to .elucidate structure-function relationships of human BHMT, and (2) to determine the mechanism(s) by which BHMT activity is modulated by nutrient- and pharmacologically induced changes in redox status, including changes in intracellular thiols, H2O2 and NO. Our collaborators and we are well prepared to perform the research outlined in this proposal. We have recently shown that human BHMT activity is regulated by thiols, H2O2 and NO in vitro, and we have obtained the crystal structure of the enzyme. The outcomes of the proposed studies will include novel information on IBHMT's structure and function and its regulation by nutritional and physiological factors influencing Iredox status.